Pilot assessment of vascular endothelial growth factor receptors and trafficking pathways in recurrent and metastatic canine subcutaneous mast cell tumours

Abstract

Canine subcutaneous mast cell tumour (scMCT) shows less aggressive biological behaviour than cutaneous MCT. Vascular endothelial growth factor receptor 2 (VEGFR2) is expressed by neoplastic cells in canine scMCT, but the relevance of this signalling pathway for disease pathobiology is not clear. The objective of this study was to quantify VEGF-A, VEGFR2, pVEGFR2, the VEGF co-receptor Neuropilin 1 (NRP-1) and the E3 ubiquitin protein ligase c-Cbl in canine scMCT, and to evaluate their association with disease outcome. Immunohistochemical staining for biomarkers was quantified from 14 cases of canine scMCT using manual and computer-assisted methods. Kaplan-Meier curves were generated for disease-free survival (DFS) and compared using Mantel-Cox log-rank analysis. Cases with high levels of neoplastic cell VEGFR2, pVEGFR2 or c-CBL immunoreactivity had significantly reduced DFS. All cases displayed neoplastic cells positive for VEGF-A, which was significantly associated with pVEGFR2 immunoreactivity. There were also significant positive correlations between VEGFR2 and pVEGFR2, and between c-CBL and pVEGFR2 levels. This pilot study demonstrates the potential utility of these markers in a subset of scMCT in dogs.

Keywords: Neuropilin‐1; VEGFR2; c‐CBL; dog.

Figure 1

Western blot detection of proteins of interest in lysates of canine MCT cell line MCT‐1; detection of α‐tubulin was used to control for protein loading.

Figure 2

Immunohistochemical detection of VEGFR2 (a), pVEGFR2 (b), VEGF‐A (c) and NRP‐1 (d) in canine subcutaneous mast cell tumours. Blood vessels positive for vascular endothelial staining of VEGFR2 and pVEGFR2 are indicated by asterisks. Nuclear immunoreactivity is indicated by black arrows and membranous immunoreactivity is indicated by green arrows. Scale bar = 50 μm. (e) Linear regression analysis of canine subcutaneous mast cell tumours reveals a significant correlation in percent positive pixels for native VEGFR2 and pVEGFR2. (f) Cases with higher VEGF‐A scores (++ and +++) had significantly more pVEGFR2 immunoreactivity. VEGFR, Vascular endothelial growth factor receptor 2; NRP‐1, Neuropilin 1.

Figure 3

(a) Immunohistochemical detection of c‐CBL in canine subcutaneous mast cell tumours. Nuclear immunoreactivity is indicated by the black arrow. Scale bar = 50 μm. (b) Linear regression analysis of canine subcutaneous mast cell tumours reveals a significant correlation in percent positive pixels for pVEGFR2 and c‐CBL. VEGFR, Vascular endothelial growth factor receptor 2.